DETERMINATION OF AN ACTIVE PRINCIPLE OF … 2017A PDF/3_Sitosterol... · their effects on diverse health disorders, e.g. antioxidant, anti-inflammatory, antibacterial and antifungal,

REVISTA BOLIVIANA DE QUÍMICAISSN 0250-5460 Rev. Bol. Quim. Paper editionISSN 2078-3949 Rev. boliv. quim. Electronic editionMartinez et al. RBQ Vol. 34, No.1, pp. 14-27, 2017

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DETERMINATION OF AN ACTIVEPRINCIPLE OF POLYGONUM

PUNCTATUM ELLIOT; FULL NMRSPECTRA OF STIGMAST-5-EN-3-OL

(500/125 MHz)

DETERMINACIÓN DE UN PRINCIPIOACTIVO DE POLYGONUM PUNCTATUMELLIOT; ESPECTROS COMPLETOS DE

RMN DE STIGMAST-5-EN-3-OL(500/125 MHZ)

Full original article

Miguel Martínez1,*, Bonifacia Benítez1, Sandra Alvarez2, Rebeca Prieto3, MónicaRolón3, Gil Valdo José Da Silva4, José A. Bravo5, José L. Vila6

1Laboratorio de Análisis de Recursos Vegetales LAREV, Área Fitoquímica, Facultad de de Ciencias Exactas yNaturales, Universidad Nacional de Asunción UNA, Av. Mariscal López 3492 c/ 26 de febrero, CampusUniversitario, phone +59521585 600/01, San Lorenzo, Central, Paraguay, [email protected]

2Laboratorio de Biotecnología, Universidad Nacional de Asunción UNA, Av. Mariscal López 3492 c/ 26 defebrero, Campus Universitario, phone +59521585562, San Lorenzo, Central, Paraguay,[email protected]

3Carrera de Biotecnología, Universidad Nacional de Asunción UNA, Av. Mariscal López 3492 c/ 26 de Febrero,Campus Universitario, phone +59521585562, San Lorenzo, Central, Paraguay

4Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de SãoPaulo USP, Av. Bandeirantes, 3900 - Vila Monte Alegre, Ribeirão Preto - SP, 14040-900, phone +55163315-3670, Brasil, [email protected]

5Laboratorio de Productos Naturales, Fitoquímica, Instituto de Investigaciones en Productos Naturales IIPN,Ciencias Químicas – Facultad de Ciencias Puras y Naturales, Universidad Mayor de San Andrés UMSA, P.O.Box 303, Calle Andrés Bello s/n, Ciudad Universitaria Cota Cota, phone +59122792238, La Paz, Bolivia,[email protected]

6Laboratorio de Productos Naturales, Síntesis y Hemisíntesis, Instituto de Investigaciones en ProductosNaturales IIPN, Ciencias Químicas - Facultad de Ciencias Puras y Naturales, Universidad Mayor de SanAndrés UMSA, P.O. Box 303, Calle Andrés Bello s/n, Ciudad Universitaria Cota Cota, phone +59122792238,La Paz, Bolivia, [email protected]

Keywords: Stigmast-5-en-3-ol, Sitosterol, Polygonum punctatum Elliott, Polygonaceae, Roots,Anti-inflammatory, NMR spectra, 500 MHz.

ABSTRACT

The aim of this report is to provide full NMR spectra (1H 500 MHz, 13C 125.8 MHz) of stigmast-5-en-3-ol (1),whose other common names are: -sitosterol, -sitosterin, 22,23-dihydrostigmasterol, and IUPAC name: 17-(5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1-cyclopenta[a]phenanthren-3-ol.,and IUPAC name: The spectra set includes HMBC, HSQC and COSY studies. ROESY or NOESY studies wereexcluded. Compound 1 is found to be the major component of the alcoholic extract of roots of Polygonum punctatum(Polygonaceae), a macrophyte native species of Paraguay with varied therapeutic applications like anti-inflammatory

Received 05 22 2017Accepted 05 23 2017Published 05 31 2017

Vol. 34, No.1, pp. 14-27, Ene./May. 201734(1), 14-27, Jan./May. 2017Bolivian Journal of Chemistry 34(1) 127-133, Jan./Apr. 2017




among others. Compound 1 was extracted by maceration in EtOH 96° and it was isolated by using chromatographictechniques. A bibliographic research allowed to establish a correlation between traditional medicine information ofanti-inflammatory property of P. punctatum and the isolated compound 1 for which a series of reports about itspharmacological properties permitted to affirm that 1, being the major secondary metabolite in the alcoholic extractof roots, is one of the active principles of the plant, namely the anti-inflammatory principle. The spectroscopic workand the structural elucidation permitted the identification of compound 1 as -sitosterol. The bibliographic researchallowed to label -sitosterol (1) as a new compound in the species P. punctatum for which only polygodial, a potentantifungal sesquiterpene, was previously described. With the present reporting of compound 1 in P. punctatum, thetotal number of secondary metabolites and active principles thus far described in the species reaches the cipher of two(according to the newest bibliographic review on the chemical content of the Polygonum genus, 2014).

*Corresponding author: [email protected]

RESUMEN

Spanish title: Determinación de un principio activo de Polygonum punctatum; espectros completos de RMN destigmast-5-en-3-ol (500/125 MHz). El objetivo de este artículo es proveer de información espectral completa destigmast-5-en-3 -ol (1), cuyos otros nombres comunes son: -sitosterol, -sitosterin, 22,23-dihydrostigmasterol, andIUPAC name: 17-(5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1-cyclopenta[a]phenanthren-3-ol., and IUPAC name: La colección de espectros incluye los estudios HMBC, HSQCand COSY. Los estudios ROESY o NOESY no están contemplados. El compuesto 1 resultó ser el componentemayoritario del extracto alcohólico de Polygonum punctatum (Polygonaceae), especie macrófita nativa de Paraguaycon aplicaciones terapéuticas como: hemorroides, abortos y otras. El compuesto 1 fue extraído por maceración enetanol 96° y aislado mediante la aplicación de técnicas cromatográficas. Una investigación bibliográfica permitióestablecer una correlación entre la información de la medicina tradicional de la propiedad antiinflamatoria de P.punctatum y el compuesto aislado 1 para el cual una serie de informes sobre sus propiedades farmacológicas permitióafirmar que 1, siendo el componente mayoritario del extracto alcohólico de las raices, es uno de los principios activosde la planta, es decir, el principio anti-inflamatorio. El trabajo espectroscópico y la elucidación estructuralcondujeron a la identificación del compuesto 1 como -sitosterol. La investigación bibliográfica permitió añadir -sitosterol (1) como un nuevo compuesto en la especie Polygonum punctatum para la cual solo se describiópreviamente un sesquiterpeno antifúngico potente, polygodial. Con la presente descripción del compuesto 1 en P.punctatum, el número total de metabolitos secundarios y principios activos hasta ahora descritos en la especie alcanzala cifra de dos (según la última revisión bibliográfica sobre el contenido químico del género Polygonum, 2014).

INTRODUCTION

Polygonum genus review

Various species of the genus Polygonum (Polygonaceae) are of common use in traditional Chinese medicine due totheir effects on diverse health disorders, e.g. antioxidant, anti-inflammatory, antibacterial and antifungal, anticancer,antiviral, lipid-regulating, neuroprotective and the estrogenic effect [1]. Thirty-four different Polygonum species havebeen reported as the source for different traditional medicine uses in the eastern Himalayan region, India [2]. Theplant remedies are used as astringent, laxative, febrifuge, demulcent, tonic, diuretic, gonorrhea relief, relief ofmenstrual and other gynecological problems, dysentery relief, chest related problems relief, wound healing agents,jaundice relief, piles relief, muscular pain relief, cardiac problems relief, colic relief [2]. As Polygonum punctatumElliott is endemic in the three Americas, it is traditionally used in Paraguay for sore throats and colds (anti-inflammatory) [3]. Important discoveries in the treatment of various illnesses like for instance those related to snake-bites, inflammatory problems, cancer, bacteria, viral infections, antiseptic, fungal, diabetic, hypertension,hyperlipemia, jaundice, hemorrhage, cough, fever, ulcers, skin affections, anemia, diarrhea and various urologicdisorders, have been exposed in ethnobotanical informs and some of them have been in some degree proved byexperimental approaches on ethanol extracts of some Polygonum species [4]. For example, Polygonum cuspidatum isuseful as analgesic, antipyretic, diuretic, and expectorant as well as for the healing of arthralgia, chronic bronchitis,jaundice, amenorrhea and high blood pressure, according to Chinese traditional medicine [5]. Polygonum cuspidatumhas been evaluated with respect to its antioxidant capacity [6,7] as well as for example the anti-inflammatory activityas inhibitor of NF-kb [8,9,10]. The antinociceptive, anti-inflammatory and diuretic properties of Polygonum




barbatum (L.) have been assessed on the mice/rat model; the plant was collected in Bangladesh [11]. Thethrombolytic activity, the membrane stabilizing activity and the cytotoxic activity of Polygonum hydropiper wasevaluated. The thrombolytic activity, the membrane stabilizing activity were assessed by means of the use of humanerythrocytes, the results were compared with standard streptokinase and acetyl salycilic acid as standard anti-inflammatory respectively. Citotoxicity was established by Brine shrimp lethality bioassay. The plant was providedfrom Bangladesh. Phytochemical screening revealed the presence of saponines, steroids, flavonoids andcarbohydrates in extracts derived from the applying of organic solvents of increasing polarity to the plant material[12]. Polygonum hydropiperoides and P. spectabile traditionally employed by their medicinal properties namely anti-inflammatory, antihaemorrhoidal and anti-diarrhea have been studied under a pharmacognostic scope. Thepharmacognosy screening approach employed revealed the presence of triterpenes and/or steroids, coumarins,flavonoids, polyphenols, tannins and saponins. The plant samples were collected in Minas Gerais, Brazil [13].Polygonum hydropiperoides also showed activity against Staphylococcus aureus, confirming the traditional use ofthe leaves of the plant against bacteria; plant samples were collect in northern Peru [14]. The leaves of Polygonumpersicaria find application as astringent, rubefacient and vermifuge [15,16], the hydromethanolic extract of the leavesmanifested good activity against Staphylococcus aureus and Enterobacter aerogenes [15]. Polygonum plebejum isused in case of pneumonia [15,17,18]; a good activity was found for the hydrodromethanolic extract againstPseudomonas aeruginosa and Enterobacter aerogenes [15]. Polygonum persicaria and P. plebejum showedimportant insecticidal activity against Tribolium castaneum [15]. Polygonum cognatum showed activity againstStaphylococcus aureus and Bacillus subtilis [15,19]. Quercetin from Polygonum equisetiforme is the responsible forthe narrowed spectrum and high activity against Citrobacter frundii, Escherichia coli, Enterobacter aerogenes,Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa [15,20]. Polygonum chinense has beenevaluated for its antifungal activity [15,21]. Polygonum persicaria was investigated due to its ethnopharmacologicalinformation regarding an antifungal activity; polygodial showed good activity against yeasts and dermatophytes [22].The Bangladeshi medicinal plant Polygonum stagninum Buch.-Ham. ex Meissn. was evaluated due to analgesic andanti-inflammatory properties by examining its extracts of n-hexane, ethyl acetate (EtOAc), and methanol of the aerialparts [23].

Polygonum punctatum review

Polygonum punctatum (Pp) known in Brazil as “erva-de-bicho”, is ethno-pharmacologically described for thetreatment of intestinal disorders [15,24]. Also, the dichloromethane extract of the aerial parts of Pp manifested strongactivity in a bioautographic assay over the fungus Cladosporium sphaerospermum; the procedure afforded thesesquiterpene dialdehyde: polygodial as the antifungal active principle of the dichloromethane extract [15,24]. For acomplete approach to the pharmacological properties of polygodial see reference [24] and related references therein.Pp is mentioned as an anti-inflammatory and diuretic in traditional medicine treatments [11]. According to Choudaryet al., the leaves of Pp are employed for swelling treatments [2]. Pp is used as antifungal, antihistaminic, anti-inflammatory and hypotensive [4,24,25]. Pp was included as part of a study carried out to show the importance ofnon-arboreal native plants in maintaining a rich diversity of bees in Santa Catarina island, southern Brazil [26].According to the authors, shoots of Pp are used to treat varicose veins and varicose ulcers. The plant, together withother 58 Brazilian plants, was analyzed with respect to its contents in diverse metals by neutron activation analysisand atomic absorption [27]. The antibacterial (Gram+ and Gram-) and antifungal activity was evaluated for nativespecies of Argentina including Pp; the 50% hydroethanolic extract of the aerial parts was used as starting vegetalmaterial [28]. A wide antimicrobial activity of the dichloromethane and methanol extracts of the aerial parts of Ppwas found: The dichloromethane extract was active against Bacillus subtillis, Micrococcus luteus, Listeriamonocytogenes y Staphylococcus aureus. The methanol extract was active against B. subtilis, M. luteus. L.monocytogenes y S. aureus. This same extract was active against Aspergillus níger. The dicloromethane extract wasactive against Candida albicans y A. niger [29]. The good anti-inflammatory activity pf Pp was established byCarrageenan-induced pedal edema/gastric intubation in rats [25,30]. Pp was reported as a plant for healing wounds inBrazil [31]. -Bisabolene is the major monoterpene component of the essential oil of Pp; the anti-inflammatoryethnopharmacological property as well as the antifungal and antibacterial activity of its organic extracts is mentioned[28,32,33]. The mutagenic and genotoxic potential of Pp was evaluated; infusions and extracts of leaves showedreduced values of mitotic indices in the concentrations used compared to the control in distilled water. Chromosomalalterations were identified in the cell division in all concentrations of infusions and extracts, demonstrating that theplant possesses antiproliferative and genotoxic properties [34].




RESULTS AND DISCUSSION

Given the fact of plenty of references allusive to the ethnopharmacological medicinal properties of the aerial parts ofPolygonum punctatum used under different forms, and that to the best of our knowledge the roots of the species havenot yet been studied, we have carried out the phytochemical approach to this organ in the species. Moreover, let’ssignal at this point, that according to the most recent phytochemical bibliographic review on the genus [4], onlypolygodial has been thus far reported in the aerial parts of the species [24]. We contribute in the present work withthe presence of -sitosterol in the roots of the species as the major metabolite. The plant was collected inDepartamento Central, Paraguay, and the treatment of the roots included the extraction by ethanol 96% solvent andthe isolation of the major compound -sitosterol (1) by chromatographic means. The identification of 1 was possiblethanks to the application of NMR techniques.

Succinct spectral/structural heteronuclear remote correlations of 1 by NMR

The 1H NMR spectrum of 1 (Fig. 1) shows aliphatic signals between 2.50 and 0.65; this spectral zonecomprehends methyl and methylene protons. At 3.51 ppm a multiplet corresponding to a methine and at 5.35 adouble doublet corresponding to a vinyl proton. The concomitant analysis of the 13C NMR (Fig. 2) and the DEPT-135 spectra of 1, (Fig. 3) resulted in the establishment of 3 quaternaries, 9 methines, 11 methylene, and 6 methylgroups for a total of 29 carbons. The analysis of the HMBC spectrum of 1 (Fig. 4) conducts to the establishment ofremote heteronuclear correlations for the protons of the seven methyl groups as follows. Protons at 0.68 (H-18) areremotely coupled to carbons at 39.8 ppm (C-12), 42.3 ppm (C-13) and 56.8 ppm (C-14). The methyl protons at0.85 (H-27) are correlated to carbons at 28.3 (C-25), 45.8 (C-24) and 19.4 (C-26). The methyl protons at1.02 (H-19) are connected to carbons at 140.7 (C-5) 50.1 (C-9) and 36.5 (C-10). The methyl at 0.90 (H-21)can be correlated to 56.0 (C-17), 36.2 (C-20) and 33.9 (C-22). The methine proton 3.52 (H-3) can becorrelated to the quaternary 140.7 (C-5) and the methylene at 31.91 (C-2).

Table 1. 13C NMR of compound 1Atom H[35]

ppmC 1ppm

C[35]

ppmDEPT-135

1234567891011121314151617181920212223242526272829

1.471.563.522.28-5.362.031.671.48-1.521.49-1.501.601.841.490.681.021.640.940.881.041.501.650.830.851.040.88

37.2431.9171.8242.32140.74121.7331.6631.9050.1136.5021.0839.8042.3056.7624.3129.1256.0411.8619.8336.1518.7833.9326.0345.8228.2519.4019.0223.0511.98

37.2831.6971.8242.33140.70121.7231.6931.9350.1736.5221.1039.8042.3356.7924.3728.2556.0911.8619.4036.5218.7933.9826.1445.8828.9119.8018.7923.1011.99

CH2

CH2

CHCH2

CCHCH2

CHCHCCH2

CH2

CCHCH2

CH2

CHCH3

CH3

CHCH3

CH2

CH2

CHCHCH3

CH3

CH2

CH3

Figure 5. HMBC correlations of compound 1, stigmast-5-en-3-ol

12

3

54 67

89

10

1711 12 13

14

15 16

18 20

19

2122

232425

26

26

27

28

29

The methylene protons resonating at 2.0 (H-7) findcorrelation with carbons at 56.8 (C-14) and 50.1 (C-9), .The methyl protons at 0.88 (H-29) correlate to the methine at 45.82 (C-24) and with carbon 23.05C-28). Proton 5.35(H-6) can be correlated to carbons at 31.7 ppm (C-7), 36.5 ppm(C-10) and 42.3 ppm (C-4). These correlations are graphicallyexposed in Fig. 5. The established structure corresponds tostigmas-5-en-3-ol according to the comparison of 13Cchemical shift values of 1 and -sitosterol previously published[35] in Table 1.




The full establishment of the planar structure of 1 was achieved not only on the basis of the analysis of the HMBCspectrum (Fig. 4) but also by the complementary using of the HSQC (Fig. 6) and the COSY spectrum (Fig. 7).

Figure 1. 1H NMR spectrum of 1, 500 MHz, CDCl3, values measured from TMS,




Figure 2. 13C NMR spectrum of 1, 125 MHz, CDCl3, values measured from TMS




Figure 3. DEPT-135 spectrum of 1

Figure 3. DEPT-135 experiment of compound 1




Figure 4. The HMBC spectrum of compound 1




Figure 6. The HSQC spectrum of compound 1




Figure 7. The COSY spectrum of compound 1

From Fig. 5 and Table 1 it’s possible to notice that following the colors that agglutinate proton and carbons in sevendifferent groups (seven colors) and an eighth group (underlined in Table 1), from 29 carbons there are only sixgroups: CH2-1, CH-8, CH2-11, CH2-15, CH2-16 and CH3-23, that have not been correlated in any form by theHMBC experiment (they’re neither colored nor underlined); this shows the high potential of the technique HMBC inconnecting atoms in the inside of different structural portions of the global structure as well as between pairs ofpartial structures in order to correlate them. Complementarily, structural information can be derived from spectraldata by means of the analysis of the homonuclear vicinal and geminal couplings exhibited by the COSY experiment(Fig. 7). The fundamental spectrum HSQC (Fig. 6) permits the ultimate completion of Table 1 with respect to everychemical shift value in protons as well as in the carbon frequency.

EXPERIMENTAL




General

NMR experiments were performed at room temperature (298 K) on a Bruker DRX 500 spectrometer (B0 11.7 T)equipped with a mm inverse multinuclear probe (500.13 MHz for 1H and 125.77 MHz for 13C). NMR data wereprocessed using TopSpin software by Bruker BioSpin; chemical shifts were referenced to TMS as internal standard.Sample for NMR analyses was dissolved in CDCl3 in a 5 mm NMR tube. Ethanol 96% of commercial origin and nofurther purified. Chromatographic tools can be characterized as follows. Aluminum TLC plates coated with silica gel60 F254, 20X20 cm, 250 μm by MERCK. Revealing agent: H2SO4 30 % aqueous solution plus heating. LC opencolumn: silica gel 60 G, 0.063-0.200 mm, 70-230 Mesh ASTM by SCHARLAU, LC open column solvents bySIGMA ALDRICH: hexane p.a., ethyl acetate p.a. Other equipment: BÜCHI rotavapor R-200 and heating bath B-490 plus diaphragm vacuum pump ILMVAC.

Plant material

Polygonum punctatum Elliot, in Sketch Bot. S. Carolina [Elliott] 1: 455. 1817 [Dec 1817]. Syn.: Polygonumacre Kunth, hom. illeg., Polygonum epilobioides Wedd., Polygonum hydropiperoides Pursh, hom. Illeg., wascollected at compañía Piquete Cué Limpio City, Departamento Central, Republic of Paraguay in summer 2012. TheGPS coordinates were: 25°19'39.26"S, 57°25'26.43"O. A voucher specimen under the code MMNº:09 was depositedat FACEN Herbarium, School of Natural and Exact Sciences of Universidad Nacional de Asunción. The taxonomicidentification was done by one of us (B. Benítez) through the use of the key of Cialdella & Brandbyge (Flora delParaguay – 33- Polygonaceae. Conservatoire et Jardin botaniques de Genève de la Ville de Genève & MissouriBotanical Garden. pp. 106. 2001). The nomenclature was verified in Tropicos (Tropicos.org.Missouri BotanicalGarden. 22 May 2017. In: http://www.tropicos.org/Name/26000886, 2017) and in Flora del Cono Sur (2016), andThe Plant List (2015).

Extraction and isolation of 1

800 g of ground dry roots were macerated in 3.3 L of ethanol 96% thrice (10 L in total) during one week. The threemacerates were added in one to sum up a total of 7 L of ethanol extract. The extract was driven to dryness byelimination of solvent under reduced pressure. The ethanol dry extract weighed 280 g (35% yield with respect tostarting material). 1 g of dry ethanolic extract of roots was chromatographed in a LC open column using 10 g of silicagel as stationary phase and a binary mixture of the hexane-ethyl acetate system (hex/EA). Various volumes of thebinary mixture hex/EA were prepared: (100/0, 100 mL), (95/5, 100 mL), (90/10, 100 mL), (85/15, 100 mL), (80/20,100 mL), (70/30, 100 mL), (0/100, 100 mL). 205 fractions of 15 mL each were collected. The composition offractions and the increment of polarity of the binary mixture added was monitored by TLC plates using hexane pure,and a gradient of hex/EA as eluent of plates. The 205 fractions were regrouped in 13 new fractions according to TLCanalyses. The 13 fractions were analyzed in a general view in analytical TLC plate using hex/EA 70/30. Each newfraction was evaporated at reduced pressure and weighed. Fr1: 1-8 (90 mg), Fr2: 8-21 (110 mg), Fr3, 22-52 (420mg), Fr4 (130 mg), 53-64 (44 mg), Fr5: 65-77 (24 mg), Fr6: 78-83 (32 mg), Fr7: 84-88 (65 mg), Fr8: 89-97 (127mg), Fr9: 98-101 (12 mg), Fr10: 102-153 (12 mg), Fr11: 154-187 (13 mg): Fr12: 188-198 (9 mg), Fr13: 200-205 (3mg). From TLC analyses, Fr3 was chosen for further purification. Dissolved in hex/EA, it was put in refrigerator (-4°C) overnight. White crystals appeared and filtered to recrystallize in ethanol. The NMR analyses of the crystalsrevealed compound 1. Final weight of 1 was 125 mg (12.5 % by 1 g of extract, 0.047 % of extract, 0.02 % of roots ofthe plant). Fr2 and Fr4 contained only traces of compound 1.

Spectral data of compound 1

For 13C NMR data of compound 1 see Table 1.

CONCLUSIONS

We have boarded the phytochemical approach of the roots of Polygonum punctatum Elliot being this the first reporton the subject. We have reported -sitosterol for the first time in the plant and particularly in the roots of the species.The other only metabolite previously described was the antifungal polygodial [24]. -sitosterol, although verycommon in the vegetal kingdom, is a very important natural product. Plenty of uses are attributed to the compound, it




has been the object of many patents and the bibliography regarding its varied pharmacological applications is vast. Arecent review (2014) on the properties of -sitosterol mentions its characteristics as firstly and most important anti-inflammatory, but also as apoptosis inducer, chemopropective and chemopreventive, hypocholesterolemic,angiogenic, analgesic, anthelmintic and antimutagenic, immunomodulatory, partial anticancer in prostate, antioxidant,neuroprotector, antidiabetic, larvicidal, neutralizer of cobra venom, antifungal, antibacterial, and trypanocidal [36].The absorption of -sitosterol by the digestive track is weak [36], hence, -sitosterol alone, under the focus of a puremedicine has not the desired efficacy as when contained in an herbal preparation, as for instance a plant extract [36].Therefore, the traditional medicine reference to the use of plants containing the compound becomes important whendetermining its biological activities. There is a large bibliography containing information about the anti-inflammatoryproperties of -sitosterol; we mention here only a few, all these studies clearly establish the anti-inflammatorypotential of -sitosterol when used alone or in conjoint with other substances [37-44]. When studying Cyperusrotundus, -sitosterol demonstrated to possess a potent anti-inflammatory activity comparable to that ofhydrocortisone and oxyphenbutazone via intraperitoneal. -sitosterol also possessed antipyretic activity similar tothat of aspirin [37]. The low toxicity of -sitosterol was evidenced by its LD50 that was more than 3 g/kg i.p. in miceand minimum ulcerogenic dose was 600 mg/kg i.p. in rats [37]. During the approach to the study of Opuntia ficus-indica (cactus) and thanks to previous results revealing a potent anti-inflammatory activity in the ethanol extract ofstems of the species, the methanol extract was fractionated and -sitosterol isolated and evaluated to be the anti-inflamatory principle of the methanol extract of cactus stems [38].-sitosterol exhibited anti-inflammatory activity inhuman aortic endothelial cells [39]. The species Sideritis foetens Clem was surveyed on its sterols content with theobtaining of a fraction showing campesterol (7.6%), stigmasterol (28.4%) and -sitosterol (61.1%). This mixturewhere the major component was -sitosterol (61.1%) manifested anti-inflammatory activity in in vivo murine modelsof inflammation [40]. Results obtained from the anti-inflammatory assays on the extracts of leaves of the Indianmedicinal plant Nychtanthes arbortristis showed that β-sitosterol (5, 10 and 20 mg/kg, i.p.) was responsible for thesignificant and dose-dependent anti-inflammatory activity concluding thus that the compound is the responsible of theanti-inflammatory activity ascribed to the plant in India [41]. It has been disclosed that β-sitosterol in conjoint withphenolics (aloenin, barbaloin and aloe-enodin) and fatty primary alcohols (26, 28, 30 and 32 carbons) as a synergicadduct were playing an important role in the anti-inflammatory attribute of Aloe arborescens Miller [42]. Oxalisorniculata is a plant used as pain relief and with anti-inflammatory properties according to traditional medicine inIndia. -sitosterol, isolated from the leaves of the species showed to be responsible for the analgesic and anti-inflammatory activity in the leaves; the actions were manifested through central mechanism [43]. Culcasia scandensP. Beauv, a well-reputed plant for its anti-inflammatory qualities, was investigated. The results of the bio-guidedseparation of the methanol extract of the leaves from which fraction C was obtained as the most active, indicated thatthe anti-inflammatory principle was β-sitosterol [44].

We have already reviewed in the introduction section of the present article some of the Polygonum specieswhich possess anti-inflammatory properties as traditional medicine remedies [6,7,11-13,23]. Polygonum punctatumwas also described in the literature for its anti-inflammatory properties when used as a traditional remedy [4,11,24,25,30]. The part of the plant attributable to the anti-inflammatory property in the cited bibliography is usually theaerial parts or the leaves.

This bibliographic research serves to conclude firstly, that Polygonum punctatum is used as a traditionalmedicine due to its anti-inflammatory properties. Second, the aerial parts or the leaves are commonly used in gallenicpreparations from the plant itself. Third, it is feasible that the anti-inflammatory property of the plant is due to thepresence of polygodial in the aerial parts, however this compound is in reality responsible of the antifungal activity[24]. Fourth, the presence of β-sitosterol in roots has been proved in the present paper. β-sitosterol is a potent anti-inflammatory principle. Fifth, since the aerial parts (or leaves) of Polygonum punctatum are used as anti-inflammatory, and given the fact of the important quantity of β-sitosterol currently found in the roots, we propose β-sitosterol as the responsible of the anti-inflammatory principle in the aerial parts of Polygonum punctatum.

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